Matrix-assisted laser desorption and ionization (MALDI) has developed into an important tool for the analysis of numerous compositions, especially complex biological materials. MALDI uses a chemical matrix to suspend and retain one or more analytes prior to subjecting the matrix and analytes to laser desorption and ionization, typically during mass spectrometry. Prior to the development of current organic matrices used in MALDI, it was difficult to ionize intact analyte molecules without molecular fragmentation.
Numerous matrices have been developed over the years to fulfill the poorly understood requirements for successful laser absorbtion and analyte ionization without fragmentation of the analyte. The use of these matrices has become important because they have permitted the analysis of organic compositions that would otherwise not be readily observable using laser desorption and ionization methods.
MALDI has been successfully used to identify peptides, proteins, synthetic polymers, oligonucleotides, carbohydrates, and other large molecules. Unfortunately, traditional MALDI has drawbacks for the analysis of many small molecules because signals from the chemical matrix interfere with signals from analyte molecules. FIGS. 1 and 2 show spectra of two common matrices, 2,5-Dihydroxy-benzoic acid (DHBA) and Alpha Cyano-4-hydroxy-cinnamic acid (α-CHCA). These spectra show numerous peaks that potentially interfere with analysis of the mass spectra of other materials.
Chemical matrices have many other undesirable consequences besides signal interference. For example, matrices can complicate sample preparation, and the additional processing steps and materials risk the introduction of contaminants into the sample. Both the matrix and analyte must typically be dissolvable in the same solvent, further complicating sample preparation. The matrix can also make it more difficult to interface separation techniques, and inhomogeneous sample spots can lead to a sweet-spot phenomenon wherein higher amounts of analyte and matrix crystals aggregate along the perimeter of the sample drop, leading to reduced reproducibility of spectra.
The co-crystallization process of sample and matrix is also often harsh, risking the denaturation or aggregation of proteins. Additionally, it is not always clear which matrix is appropriate for a given sample. For example, matrices that are effective for peptides and proteins often do not work for oligonucleotides or polymers. Furthermore, different matrices may be required in the positive-ion detection mode and the negative-ion detection mode. Thus, an exhaustive trial and error search can be required to find the optimal matrix.
Another difficulty with MALDI is that the currently used desorption substrates are typically metal plates. These metal plates are expensive and they typically must be cleaned after use so that they can be reused. Cleaning the metal plates is time consuming and presents the possibility of carryover contamination, and also does not allow for using the substrate as a storage device for archiving the analyte samples for additional analysis. Therefore, a need exists for a method and apparatus for reducing or eliminating the need for matrices.
In 1999, a matrix-free method was described by Wei et al. in U.S. Pat. No. 6,288,390. Wei discloses the use of silicon wafers that have been electrochemically etched with an HF/ethanol solution under illumination and constant current. The sample, in solvent, is applied directly to the silicon without the addition of any matrix. This new method, labeled desorption/ionization on silicon (DIOS), allowed for the ionization of molecules within the mass range of 100 to 6000 Da without the interference caused by a matrix. Some spectra obtained using DIOS, however, have been difficult to reproduce, and the shelf life of the DIOS chips is often short. Also, DIOS chips are relatively expensive due to the high cost of the materials and processes used in their manufacture.
Therefore, a need remains for an apparatus and method that provides enhanced laser desorption in comparison to conventionally used techniques. There is also a need for an analyte desorption substrate that is sufficiently inexpensive so that it can be used and then discarded or archived.